Rigorously controlled gene expression is essential for cellular proliferation and differentiation. In recent years, more and more evidence show that the 3D organization of the genome is important for regulation of gene activity. Chromosome Conformation Capture (3C) technique and its derivatives (4C, Hi-C, etc.) have revealed extensive intra- and inter-chromosomal interactions in various eukaryotic species. Some of these interactions, such as the enhancer-promoter looping at the beta-globin Locus Control Region, play an essential role in gene regulation. Another type of long-distance interaction, homologous pairing, can also affect gene expression in somatic diploid cells. Both long-distance chromosomal interactions and homologous pairing were observed in budding yeast during vegetative growth. However, the functional significance of these interactions are not understood. The goal of this proposal is to identify and mechanistically dissect long-distance interactions tha regulate gene expression in budding yeast. We will assay gene expression in single live cells to probe the regulatory effect on the average level, cell-to-cell variability (noise), and dynamics of gene expression. In Aim 1, we focus on gene regulatory effect by interactions between homologous alleles. Our preliminary data have provided strong evidence that a reporter gene can make contacts with its allelic copy and influence its activity. We plan to fully characterize this phenomenon and elucidate the underlying mechanism. In addition, we will pioneer a method to manipulate long-distance chromosomal interactions and probe the corresponding effect on gene regulation. In Aim 2, we will study gene regulation by non-homologous long-distance chromosomal interactions. We designed a novel yeast library containing dual reporters to screen for long-distance interactions that lead to change in gene expression. Altered reporter expression (both the average level and noise) was found in a fraction of the library strains, potentially caused by chromosomal looping / clustering. We propose further characterization of the gene regulation in these strains and an in-depth analysis of the regulatory mechanism. Completion of these studies will provide key insights into the regulatory function of long-distance interactions and establish budding yeast as an important model for studying long-distance gene regulation. These works may also generate proof-of-principle concepts for designing similar experiments in multi-cellular eukaryote.